The major goal of this proposal is to determine the role(s) of the DNase II endonuclease in phagocytosis, development and microbial infection. In previous work, we identified and purified a mammalian endonuclease activity based on its strong cleavage preference for immunoglobulin isotype-switch recombination sequences. Homology searches with peptide sequences derived from the purified mammalian protein revealed significant similarities to predicted mammalian, nematode and fruit fly proteins. Based on this information, we cloned the corresponding murine and human genes, which were subsequently found to encode DNase II, an enzyme implicated in phagocyte-mediated DNA degradation. In recent work, we cloned and characterized the homologous gene in the nematode C. elegans, demonstrating that it encodes an acid endonuclease with significant sequence and biochemical similarity to mammalian DNase II. Furthermore, we demonstrated that this acid endonuclease activity was, in fact, the NUC-1 nuclease previously implicated in the C. elegans cell death pathway. The identification of NUC-1 as a DNase II homologue was the first demonstration that DNase II enzymes are essential for the clearance of apoptotic DNA. During the past year, we have cloned and characterized a homologous ORF (CG7780) from Drosophila melanogaster. Our recent work revealed that wild-type fruit flies contain a potent acid endonuclease activity with cleavage preferences similar to DNase II/NUC-1. Analysis of extracts derived from an available acid-DNase mutant line revealed a repressed nuclease activity exhibiting altered DNA cleavage preference. Furthermore, sequence analysis of the CG7780 ORF from the acid-DNase mutant line revealed that the altered nuclease activity was caused by a point mutation within its coding region. Further confirming that CG7780 encodes the Drosophila orthologue of DNase II/NUC-1, transgenic flies expressing wild-type CG7780 ORF were subsequently shown to complement the acid-DNase mutant phenotype. As described in this proposal, we intend to perform loss-of-function experiments in two model organisms, mice and Drosophila fruit flies, to conclusively determine the in vivo role(s) of this enzyme. This study should greatly enhance our current understanding of the molecular and biochemical roles of an enzyme that has been implicated in programmed cell death and engulfment-mediated DNA degradation.